Bacteria and culture conditions.
L. monocytogenes strains EGDe (clinical isolate, serovar 1/2a), WSLC 1001 (ATCC 19112, serovar 1/2c), WSLC 1042 (ATCC 23074, serovar 4b), Scott A (clinical isolate, serovar 4b), and WSLC 1363 (soft cheese isolate, serovar 4b); L. innocua WSLC 2012 (ATCC 33091, serovar 6a); and L. ivanovii WSLC 3009 (clinical isolate, serovar 5) were grown in 0.5× BHI medium (Oxoid) for 16 to 20 h at 37°C. Cultures were diluted in phosphate-buffered saline (PBS; 120 mM NaCl, 50 mM NaH2PO4, pH 8.0) containing 0.1% Tween 20 (PBST) to the required cell densities.
(WS 3080), Pseudomonas fluorescens
(WS 1760), and Enterococcus faecalis
(WS 1761) were grown in PC (plate count) broth (Merck) at 30°C; Staphylococcus aureus
(WS 1759), B. cereus
(HER1399), and Escherichia coli
(WS 1333) were grown in PC broth at 37°C; Lactobacillus brevis
(WS 2120) was grown in MRS broth (Oxoid) at 37°C under anaerobic conditions; and Lactococcus garvieae
(WS1029) was grown in M-17 broth (Oxoid) at 30°C under anaerobic conditions. C. perfringens
ATCC 3626 was cultivated in TGY medium at 35°C in an anaerobe chamber as previously described (33
Standard plating procedure for detection of listeriae.
Detection of listeriae was performed as described in IDF standard 143A:1995 (for milk and dairy products) and ISO norm 11290-1 (for all other foods). In general, samples of 25 g each were first homogenized in a stomacher blender (Seward, Norfolk, United Kingdom), if applicable. In the IDF procedure, 50 ml of citrate buffer (50 mM sodium citrate, pH 7.4) was used for homogenization and then added to 175 ml of selective Listeria ANC enrichment broth (Merck, Darmstadt, Germany). In the ISO protocol, samples are homogenized directly in 225 ml of half-strength Fraser enrichment broth (Merck). After incubation at 30°C for 24 and 48 h, 1 loopful of the enrichment culture was streaked onto selective Oxford Listeria agar (Merck). Typical Listeria colonies were enumerated after an additional 24 h and 48 h of incubation of the plates at 37°C.
Recombinant proteins consisting of six-His-tagged N-terminal green fluorescent protein fused to C-terminal CBD118 or CBD500 were produced in E. coli
and purified as previously described (15
). Purified proteins were adjusted to 2.5 mg/ml in PBS buffer and stored at −20°C.
Coating of paramagnetic beads.
Ni-nitrilotriacetic acid (NTA) agarose beads (QIAGEN, Hilden, Germany) feature Ni-NTA groups on their surface suitable for binding of six-His tags. Aliquots of 100 μl were coated by mixing with 30 μl of CBD500 for 20 min in a horizontal shaker at 900 rpm. The beads were then separated with a magnetic stand (MPC-S; Dynal) and washed twice with 0.5 ml of buffer A (0.5 M NaCl, 0.05 M Na2HPO4, 5 mM imidazole, pH 8.0) and twice with buffer B (buffer A with 25 mM imidazole). Beads were resuspended in PBS (approximately 4.5 × 106 beads/ml) and stored at 4°C.
Lyophilized M-270 Epoxy Dynabeads (Dynal, Oslo, Norway) were suspended in diethyleneglycol-dimethyl ether to a final concentration of 30 mg/ml as recommended by the manufacturer. For coating, 400 μl of beads was washed twice with 800 μl of PBS and resuspended in 100 μl of PBS and 200 μl of 3 M (NH4)2SO4 (pH 7.4). A 100-μl volume of CBD118 or CBD500 was then added, and mixtures were incubated in an overhead rotator at 4°C for 16 h at 10 rpm and then incubated at ambient temperature (22°C) for 6 h. Residual epoxy groups were blocked by washing the beads four times with either PBS-BSA buffer (PBS containing 0.1% bovine serum albumin, pH 7.4) or Tris buffer (1.0 M Tris, pH 7.4). The CBD-coated beads (2.0 × 109 beads/ml) were stored at 4°C either in PBS-BSA or in Tris buffer containing 0.02% NaN3.
The influence of the blocking agent on the binding and recovery by CBD-coated Dynabeads was tested. Separate batches of CBD500- or CBD118-coated beads were blocked with either BSA or Tris, respectively. Different concentrations (1.0 × 107 to 4.0 × 107) of beads were incubated with 104 Listeria cells for 40 min, and the separation efficiency was determined.
Immobilization, separation, and enumeration of bacterial cells.
With the Ni-NTA agarose beads, 100-μl aliquots of Listeria cells (103 to 105 CFU/ml) were mixed with 10 to 40 μl of bead suspension (4.3 × 104 to 1.7 × 105 beads), as determined by counting of the beads in a calibrated microscope grid counting chamber (data not shown). Total volumes were adjusted to 200 μl by addition of PBST. Incubation times were 10, 20, 40, and 60 min. For detachment of cells from Ni-NTA beads, 100 μl of buffer C (0.3 M NaCl, 0.05 M Na2HPO4, 0.2 M imidazole, pH 8.0) was added, mixtures were incubated for 10 min, and supernatants containing the liberated cells were removed after magnetic separation. Following incubation of the plates at 37°C for 20 h, cells could be enumerated by colony counting.
With Dynabeads, 5- to 20-μl amounts (1.0 × 107 to 4.0 × 107 beads) were mixed with the bacterial suspensions and the tubes were rotated at 10 rpm for 10 to 40 min. After magnetic separation, beads were resuspended in 100 μl of PBST. Because cells could not be released from beads under mild conditions, the entire mixtures were plated, followed by incubation and enumeration. In order to determine the proportion of cells remaining in the supernatant and/or removed by the washing steps, supernatants were also plated.
Binding specificity of CBD118 and CBD500.
Because of their superior characteristics and performance, we carried out all further experiments with CBD-coated Dynabeads only, under the following conditions (hereafter referred to as the CBD standard procedure): 10 μl of coated beads (2 × 107 beads), 100 μl of Listeria cells (approximately 105 CFU/ml), a 40-min incubation time, and a 200-μl total volume (adjusted with PBST).
Although it has previously been shown that the two CBD species have exclusive, nonoverlapping binding ranges (15
), the two different types of CBD-coated beads were tested for cross-reactivity in a recovery assay with L. monocytogenes
EGDe and Scott A. As a negative control, unspecific binding of Listeria
cells to uncoated, Tris-blocked Dynabeads was tested.
In order to combine the different binding specificities of the CBD500 and CBD118 proteins, the following two procedures were tested, i.e., (i) beads coated with CBD118 or CBD500 were mixed in a 1:1 ratio, and (ii) CBD proteins were mixed prior to coating (mixed CBD118-CBD500 coating). Performance of the bead preparations was compared in recovery experiments as described above.
Influence of growth medium on bacterial immobilization.
To test the potential influence of the growth medium on the binding of CBD proteins to Listeria cells, strains EGDe, WSLC 1001, and Scott A were grown in BHI, Listeria ANC broth, and half-strength Fraser enrichment broth, followed by subsequent determination of recovery rates as described above.
Capture from mixed bacterial suspensions.
The ability to selectively immobilize and capture different Listeria cells (see Table ) from a mixture of various other bacteria was evaluated. For this purpose, 104 Listeria cells were mixed with approximately 104 CFU each of B. subtilis, E. faecalis, S. aureus, L. brevis, L. garvieae, P. fluorescens, and E. coli, resulting in a final ratio of Listeria cells to other bacteria of 1:7. Magnetic separation and recovery were carried out as described above. Appropriate dilutions of supernatants and resuspended beads were plated on Oxford agar plates and incubated for 24 h at 37°C.
Efficiencies of recovery of different Listeria species and serovars from mixed bacterial suspensionsa
Comparison of antibody-based IMS (anti-Listeria Dynabeads) to CBD-MS.
Commercially available anti-Listeria Dynabeads were used according to the instructions provided by the manufacturer (Dynal, Oslo, Norway). The strains used to compare their performance to that of CBD-coated beads were L. monocytogenes Scott A, EGDe, and WSLC 1001; L. ivanovii WSLC 3009; and L. innocua WSLC 2012. Recovery experiments were carried out as described above. Because of the strong agglutination tendency of the antibody-coated anti-Listeria Dynabeads, immobilization efficiency was also calculated on the basis of the number of cells remaining in the supernatant after contact with the beads.
Artificially contaminated foods.
A variety of foods frequently contaminated with listeriae were purchased at local retailers (see Tables and ). Each sample was divided into several portions of 25 g and packed into sterile plastic bags. One aliquot was then tested for Listeria contamination by the standard procedure, and the others were frozen at −80°C (if applicable). Only samples free of listeriae were used in the experiments described below.
Comparison of standard plating and magnetic separation with CBD118-coated beads for detection of L. monocytogenes EGDe in artificially contaminated food samples
Comparison of standard plating and magnetic separation with CBD500-coated beads for detection of L. monocytogenes Scott A in artificially contaminated food samples
Food samples were mixed (in the bags) with 1 ml each of appropriate dilutions of the selected test strain L. monocytogenes EGDe or Scott A to obtain initial contamination rates of 0.1, 1.0, 10, and 100 CFU/g. For the negative control, PBS buffer was added. Samples were then stored at 4°C for 24 h in order to simulate more-realistic conditions. After 6, 24, and 48 h of incubation, 100-μl aliquots of enrichment culture were removed and mixed with 10 μl of CBD-coated Dynabeads. To adjust the pH of the mixtures, 90 μl of 2× PBST was added (final volume, 200 μl). Following magnetic capture, supernatants were discarded and beads were resuspended in 100 μl of PBST, plated on Oxford agar, and incubated at 37°C for 24 to 48 h.
Validation of the CBD-MS method with naturally contaminated foods.
A diverse range of 275 food samples were purchased from local stores and supermarkets, including meat, poultry, fish, dairy products, and various ready-to-eat deli items. In this experiment, equal amounts of the two different types of CBD-coated Dynabeads (CBD500 and CBD118) were mixed prior to use. Analyses were otherwise performed as described above, including selective enrichment (24 h for CBD-MS, 48 h for the standard method) and plating on Oxford agar. Species identification was carried out by standard biochemical testing and PCR amplification of the L. monocytogenes
Analysis of results.
Viable cell counts (bead-cell complexes, cells released from beads, cells in supernatant) were determined by duplicate plating. The number of colonies was assumed to reflect the number of viable cells immobilized on beads (Dynabeads) or the number of cells released from the bead surface (Ni-NTA agarose beads). The recovery rates were calculated on the basis of these counts and the number of residual cells in the supernatant after separation and removal of beads and expressed as percent recovery. All experiments were independently performed in triplicate, results are presented as means, and standard deviations are indicated. Statistical analysis was performed with an unpaired t test and an alpha level of 0.05.
CBD proteins from Bacillus and Clostridium phage endolysins.
The CBDs from endolysins Ply21 (16
) and Ply3626 (33
) were identified by bioinformatics as previously described (15
). Briefly, C-terminal ply
gene fragments corresponding to amino acid residues K136
of Ply21 and K197
of Ply3626, respectively, were amplified and inserted into pHGFP. The HGFP-CBD21 and HGFP-CBD3626 fusion proteins were produced in E. coli
, purified by immobilized metal-chelating chromatography, and tested for the ability to fluorescently decorate cell walls of B. cereus
and C. perfringens
as described previously (15
). The organisms were cultured as specified in the paragraph on bacteria and culture conditions, and immobilization of bacterial cells on CBD-coated Dynabeads, magnetic separation, and determination of recovery rates were performed as described above for Listeria